Abstract
An inflammatory response requires leukocytes to migrate from the circulation across the vascular lining into the tissue to clear the invading pathogen. Whereas a lot of attention is focused on how leukocytes make their way through the endothelial monolayer, it is less clear how leukocytes migrate underneath the endothelium before they enter the tissue. Upon finalization of the diapedesis step, leukocytes reside in the subendothelial space and encounter endothelial focal adhesions. Using TIRF microscopy, we show that neutrophils navigate around these focal adhesions. Neutrophils recognize focal adhesions as physical obstacles and deform to get around them. Increasing the number of focal adhesions by silencing the small GTPase RhoJ slows down basolateral crawling of neutrophils. However, apical crawling and diapedesis itself are not affected by RhoJ depletion. Increasing the number of focal adhesions drastically by expressing the Rac1 GEF Tiam1 make neutrophils to avoid migrating underneath these Tiam1-expressing endothelial cells. Together, our results show that focal adhesions mark the basolateral migration path of neutrophils.
Highlights
Neutrophils in the bloodstream form the first line of defense during an infection
The mNeonGreen-Paxillin is used as a proxy for focal adhesions (FAs), enabling us to monitor in real-time FAs during neutrophil transendothelial migration (TEM) under physiological flow conditions
By using TIRF microscopy, we detected the basolateral side of the endothelial cell layer and were able to detect the FAs with high contrast in realtime
Summary
Neutrophils in the bloodstream form the first line of defense during an infection. To fulfil their function at the site of inflammation, they must exit the vasculature in a process referred to as transendothelial migration (TEM). Endothelial cells lining the blood vessels form a tight barrier involving the adherens junction protein VE-cadherin During inflammation, these junctions allow leukocytes to cross while keeping the barrier intact. Neutrophils tend to stay between the Endothelial Focal Adhesions During TEM endothelium and the basement membrane for up to 20 minutes before penetrating the inflamed tissue [3, 4]. It seems that they are actively searching for a spot to enter the tissue. Identifying these factors may provide new therapeutic options to promote or reduce tissue infiltration of immune cells
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